Ribonuclease P (RNase P) is one of only two enzymes containing catalytically active RNA subunits that are found in all living organisms. The primary known function of the enzyme is to cleave precursor tRNA transcripts to give mature 5' termini, but it is known to also have several other substrates in vivo. In eukaryotic nuclei the enzyme has evolved to be a far more complex ribonucleoprotein, having twenty times more protein than the bacterial enzyme. The evolutionary progression of this enzyme is particularly interesting, since the eukaryotic RNA subunit has lost the ability to act as a ribozyme in the absence of the protein "cofactors", even though the structure and key residues are conserved in RNA subunit. Previous studies in this lab have established the subunit composition of the Saccharomyces cerevisiae nuclear RNase P and have shown that it is part of a spatially ordered pathway for early pre-tRNA processing in the nucleus. Biochemical and genetic investigations of the RNase P subunits have revealed extensive interactions with each other and with other cellular components. Our results suggest that the large increase in subunit complexity of the nuclear RNase P relative to bacteria and organelles might be required for correct positioning and timing of RNase P cleavage in the highly ordered nuclear environment, as well as for discriminating among potential RNA substrates. Experiments described in this proposal investigate why the increased complexity of the eukaryotic RNase P holoenzyme is necessary for proper function.